This invention relates to improved methods of activation for solid phase peptide synthesis and more particularly to the use of Benzotriazolyloxytris(dimethylamino)phosphonium Hexafluorophosphate (BOP) Reagent and BOP in combination with hydroxybenzotriazole (HOBt) Modified Reagents.
Solid phase peptide synthesis typically begins with covalent attachment of the carboxyl end of a first alpha-amino protected acid through an organic linker to an insoluble synthesis resin bead. This can be illustrated as: ##STR1## wherein .circle.P is the insoluble synthesis resin, Aa.sub.1 is the first amino acid and X is a protecting group such as Fmoc, t-Boc and the like.
The general synthesis cycle then consists of deprotection of the alpha-amine group of the last amino acid, washing and, if necessary, neutralization, followed by reaction with a carboxyl activated form of the next alpha-amine protected amino acid to be added. The peptide chain then becomes: ##STR2## wherein Aa.sub.2 is the second amino acid. The cycle is repeated to the n.sup.th amino acid to yield: ##STR3## wherein Aa.sub.n is the n.sup.th amino acid.
Following the synthesis of the entire peptide chain in the desired sequence, the organic link of the peptide chain to the resin bead is cleaved and the dissolved peptide is separated from the insoluble resin and purified.
Although the process is simple in principle, it is difficult to obtain peptides having over 30 amino acids with any substantial purity. This is due to the fact that the average step yield has a profound effect on the purity of the peptide product, as illustrated by the values in the following table for synthesis of a 30 amino acid peptide.
TABLE I ______________________________________ 30 AMINO ACID PEPTIDE Step Yield Purity ______________________________________ 95.0% 21% 99.5% 86% 99.7% 91% ______________________________________
The results are even more problematic for longer peptides. A peptide with 101 residues and a step yield of 99.0% provides a product of only 36% purity. In all cases, the by-products of the peptide synthesis are a complex mixture of molecules that are chemically similar to the peptide product. Chromatographic purification is difficult and time consuming because the relative amount of by-product molecules often exceeds about 25%.
The efficiency of the step yield can be judged by the purity of the synthesized peptide and is dependent on many factors including the nature and quality of the protected amino acids, solvent purity, chemical integrity of the resin, the chemical nature of the organic linker, the form of the activated carboxyl of the amino acid, efficiency of the wash steps, the synthesis protocol, and in some instances, the identity of an amino acid in conjunction with a particular sequence segment to which it is being added. Each of these factors is significant in each coupling step.
The automated prior art coupling processes available use diisopropylcarbodiimide (DIPCDI) or dicyclohexylcarbodiimide (DCCI) as activators for addition of the next protected amino acid residue in the sequence. These activators which form symmetrical anhydride or other active intermediates work poorly in polar solvents, such as dimethylformamide. However, such solvating solvents are essential in the synthesis of large peptides because of their ability to prevent aggregation of peptide chains, and increase coupling yields.
Accordingly, it is desirable to provide an improved activation method for solid phase peptide synthesis which increases coupling efficiency and overcomes the problems associated with prior art procedures.